Abstract

Triple negative breast cancer (TNBC) comprises 15-20% of breast cancer, and carries a poor prognosis. TRAIL (Tumor Necrosis Factor-related apoptosis inducing ligand), a member of the TNF-alpha family of death receptor ligands, induces apoptosis by binding death receptors (DR4 and DR5), and can be a good therapeutic target in TNBC based on the work of Lipkowitz at the NCI. Lapatinib, a dual HER2 and EGFR inhibitor has been shown to sensitize colon cancer and GBM cells to TRAIL-induced apoptosis through an off-target effect that involves JNK. In colon cancer cell lines, this sensitization was found to be due to increased DR4 and DR5 protein levels, and this was not affected by HER2 and EGFR dual specific inhibition. The majority of breast cancer cell lines including HCC 1937, a BRCA1-deficient TNBC cell line derived from a patient with BRCA1 mutation, are known to have constitutive death receptor endocytosis resulting in resistance to TRAIL. We hypothesized that treatment of TNBC cells with lapatinib may induce sensitization to TRAIL-induced apoptosis in normally TRAIL-resistant cell lines, but possibly not by increased death receptor protein expression since these cells have defective cell surface expression. MDA-MB-231 and HCC 1937 cell lines are both well-validated TNBC cell lines. MDA-MB-231 is known to be sensitive to TRAIL, whereas HCC 1937 is resistant to TRAIL. These two cell lines were pre-treated with 0-10 μM lapatinib for 48 hrs. Cells were then treated with His-tagged recombinant TRAIL (50 ng/mL) for 24 hr subsequently. Cell Titer Glo assay, and sub-G1 DNA content analysis by flow cytometry showed increments of apoptosis after lapatinib pre-treatment. This change was more prominent in HCC 1937, which is normally resistant to TRAIL-induced apoptosis. However western blotting did not show increased DR4/DR5 by pre-treatment with lapatinib in either MDA-MB-231 or HCC 1937. This result suggests that we may have identified an off-target effect of lapatinib in TNBC that appears to not involve increased death receptor expression. We are further exploring the previously described role for JNK in the off-target effect to further elucidate this TRAIL death receptor- and Her2/EGFR-independent mechanism of TRAIL sensitization by lapatinib that has implication for TNBC therapy.